Method for enumeration of mammalian micronucleated erythrocyte populations, while distinguishing platelets and/or platelet-associated aggregates

ABSTRACT

A method for the enumeration of micronucleated erythrocyte populations while distinguishing platelet and platelet-associated aggregates involves the use of a first fluorescent labeled antibody having binding specificity for a surface marker for reticulocytes, a second fluorescent labeled antibody having binding specificity for a surface marker for platelets, and a nucleic acid staining dye that stains DNA (micronuclei) in erythrocyte populations. Because the fluorescent emission spectra of the first and second fluorescent labeled antibodies do not substantially overlap with one another or with the emission spectra of the nucleic acid staining dye, upon excitation of the labels and dye it is possible to detect the fluorescent emission and light scatter produced by the erythrocyte populations and platelets, and count the number of cells from one or more erythrocyte populations in said sample. In particular, the use of the second antibody prevents interference by platelet-associated aggregates in the scoring procedures.

This application is a continuation of U.S. patent application Ser. No.12/177,352, filed Jul. 22, 2008, which is a division of U.S. patentapplication Ser. No. 10/878,456, filed Jun. 28, 2004, now U.S. Pat. No.7,425,421 issued Sep. 16, 2008, which claims the benefit of U.S.Provisional Patent Application Ser. No. 60/482,678, filed Jun. 26, 2003,each of which is hereby incorporated by reference in its entirety.

The present invention was made with government support under grantnumber R44ES010752-02 from the National Institute of EnvironmentalHealth Sciences. The government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates generally to methods for the enumerationof micronucleated erythrocyte populations, where erythrocyte populationsare separately labeled from platelets to discriminate or preventinterference by platelets and/or platelet-associated aggregates in theenumeration thereof.

BACKGROUND OF THE INVENTION

Micronuclei (MN) are formed upon cell division in cells with DNAdouble-strand break(s) or dysfunctional mitotic spindle apparatus. Basedon this detailed understanding of MN origin, the rodent-basedmicronucleus test has become the most widely utilized in vivo system forevaluating the clastogenic and aneugenic potential of chemicals (Heddle,“A Rapid In Vivo Test for Chromosome Damage,” Mutat. Res. 18:187-190(1973); Schmid, “The Micronucleus Test,” Mutat. Res. 31:9-15 (1975);Hayashi et al., “In Vivo Rodent Erythrocyte Micronucleus Assay: Aspectsof Protocol Design Including Repeated Treatments, Integration WithToxicity Testing, and Automated Scoring,” Environ. Mol. Mutagen.35:234-252 (2000)). These rodent-based tests are most typicallyperformed as erythrocyte-based assays. Since erythroblast precursors area rapidly dividing cell population, and their nucleus is expelled a fewhours after the last mitosis, MN-associated chromatin is particularlysimple to detect in reticulocytes and normochromatic erythrocytes givenappropriate staining (e.g., acridine orange) (Hayashi et al., “AnApplication of Acridine Orange Fluorescent Staining to the MicronucleusTest,” Mutat. Res. 120:241-247 (1983)).

Target cells for erythrocyte-based micronucleus assays weretraditionally obtained from the bone marrow compartment. MacGregor etal. demonstrated that MN formed in the bone marrow of mice persist inperipheral blood (“Clastogen-induced Micronuclei in Peripheral BloodErythrocytes: The Basis of an Improved Micronucleus Test,” Environ.Mutagen. 2:509-514 (1980)). Therefore, assay sensitivity is retainedwhen studying genotoxicant-induced micronucleated erythrocytes in theperipheral blood of mice (Hayashi et al., “The Micronucleus Assay WithMouse Peripheral Blood Reticulocytes Using Acridine Orange-CoatedSlides,” Mutat. Res. 245:245-249 (1990); “Micronucleus Test With MousePeripheral Blood Erythrocytes By Acridine Orange Supravital Staining:The Summary Report of the 5th Collaborative Study by The CollaborativeStudy Group for the Micronucleus Test,” Mutat. Res. 278:83-98 (1992)).To date, peripheral blood MN studies involving species other than themouse have been qualified because it has been assumed that the highefficiency with which the spleen eliminates MN-containing erythrocytesfrom circulation would limit assay sensitivity (Schlegel and MacGregor,“The Persistence of Micronucleated Erythrocytes in the PeripheralCirculation of Normal and Splenectomized Fischer 344 Rats: Implicationsfor Cytogenetic Screening,” Mutat. Res. 127:169-174 (1984)).

Despite a historical bias against the use of peripheral blood, studieswith intact rats continue to suggest that circulating reticulocytesrepresent a suitable target population for studying genotoxicant-inducedMN [Hayashi et al., “The Micronucleus Assay Using Peripheral BloodReticulocytes from Mitomycin C- and Cyclophosphamide-treated Rats,”Mutat. Res. 278:209-213 (1992); Asanami et al., “The Suitability of RatPeripheral Blood in Subchronic Studies for the Micronucleus Assay,”Mutat. Res. 347:73-78 (1995); Wakata et al., “Evaluation of the RatMicronucleus Test with Bone Marrow and Peripheral Blood: Summary of the9th Collaborative study by CSGMT/JEMS MMS,” Environ. Mol. Mutagen.32:84-100 (1998); Abramsson-Zetterberg et al., “The Micronucleus Test inRat Erythrocytes From Bone Marrow, Spleen and Peripheral Blood: TheResponse to Low Doses of Ionizing Radiation, Cyclophosphamide andVincristine Determined by Flow Cytometry,” Mutat. Res. 423:113-124(1999); Torous et al., “Enumeration of Micronucleated Reticulocytes inRat Peripheral Blood: A Flow Cytometric Study,” Mutat. Res. 465:91-99(2000); Hamada et al., “Evaluation of the Rodent Micronucleus Assay by a28-day Treatment Protocol: Summary of the 13th Collaborative Study bythe Collaborative Study Group for the Micronucleus Test(CSGMT)/Environmental Mutagen Society of Japan (JEMS)—MammalianMutagenicity Study Group (MMS),” Environ. Mol. Mutagen. 37:93-110(2001); and Hynes et al., “The Single Laser Flow Cytometric MicronucleusTest: A Time Course Study Using Colchicines and Urethane in Rat andMouse Peripheral Blood and Acetaldehyde in Rat Peripheral Blood,”Mutagenesis 17:15-23 (2002)). For species with efficient MN-sequesteringfunction such as the rat, it has been suggested that the sensitivity ofthe endpoint is enhanced when MN analysis is restricted to the mostimmature fraction of reticulocytes, and also when the number ofreticulocytes evaluated is increased (Schlegel and MacGregor, “ThePersistence of Micronucleated Erythrocytes in the Peripheral Circulationof Normal and Splenectomized Fischer 344 Rats: Implications forCytogenetic Screening,” Mutat. Res. 127:169-174 (1984); Hayashi et al.,“The Micronucleus Assay Using Peripheral Blood Reticulocytes fromMitomycin C- and Cyclophosphamide-treated Rats,” Mutat. Res. 278:209-213(1992); Abramsson-Zetterberg et al., “The Micronucleus Test in RatErythrocytes From Bone Marrow, Spleen and Peripheral Blood: The Responseto Low Doses of Ionizing Radiation, Cyclophosphamide and VincristineDetermined by Flow Cytometry,” Mutat. Res. 423:113-124 (1999); Torous etal., “Enumeration of Micronucleated Reticulocytes in Rat PeripheralBlood: A Flow Cytometric Study,” Mutat. Res. 465:91-99 (2000); andAbramsson-Zetterberg et al., “Human Cytogenetic Biomonitoring UsingFlow-cytometric Analysis of Micronuclei in Transferrin-positive ImmaturePeripheral Blood Reticulocytes,” Environ. Mol. Mutagen. 36:22-31(2000)).

A flow cytometry-based method for simultaneously quantifying theincidence of young and mature erythrocytes, with and withoutmicronuclei, in the peripheral blood compartment of humans has beendescribed previously (Dertinger et al., “Enumeration of MicronucleatedCD71-positive Human Reticulocytes with a Single-laser Flow Cytometer,”Mutat. Res. 515:3-14 (2002)). However, it would be desirable to developa MN-assay that utilizes a nucleic acid dye with higher specificity forchromatin, is capable of higher rates of analysis, and is capable ofpreventing platelets and platelet-associated aggregates from interferingwith accurate MN measurements.

The present invention is directed to overcoming these and otherdeficiencies in the art.

SUMMARY OF THE INVENTION

A first aspect of the present invention relates to a method for theenumeration of micronucleated erythrocyte populations whiledistinguishing platelet and platelet-associated aggregates. This methodis carried out by providing a fixed sample comprising erythrocytepopulations including mature normochromatic erythrocytes (“NCE”),reticulocytes (“RET”), micronucleated normochromatic erythrocytes(“MN-NCE”), micronucleated reticulocytes (“MN-RET”), or combinationsthereof, with the erythrocyte populations being in suspension andsubstantially free of aggregates, permeable to a nucleic acid dye andRNase, with cell surface markers in a form recognizable by an antibody,and able to exhibit substantially low autofluorescence; substantiallydegrading RNA of reticulocytes in the fixed sample with RNase;contacting the fixed sample with a first fluorescent labeled antibodyhaving binding specificity for a surface marker for reticulocytes andwith a second fluorescent labeled antibody having binding specificityfor a surface marker for platelets, wherein the fluorescent emissionspectrum of the first and second fluorescent labeled antibodies do notsubstantially overlap; staining cellular DNA with a nucleic acidstaining dye having a fluorescent emission spectrum which does notsubstantially overlap with the fluorescent emission spectrum of thefirst and second fluorescent labeled antibodies; exciting the nucleicacid staining dye, the fluorescent label associated with the RET, andthe fluorescent label associated with platelets using light ofappropriate excitation wavelength for both the nucleic acid staining dyeand the fluorescent labels to produce fluorescent emission; anddetecting the fluorescent emission and light scatter produced by theerythrocyte populations and platelets, and counting the number of cellsfrom one or more erythrocyte populations in said sample.

According to one approach, each cell in the sample is counted and thetotal number of each population (e.g., NCE, RET, MN-NCE, MN-RET) isdetermined.

According to another approach, only RET labeled with the firstfluorescent labeled antibody are counted. This is particularly usefulwhen a concentrated cell sample is utilized, there precluding the needto count the vast majority of cells (i.e., NCE) present in the sample.This approach affords a significant faster scoring procedure, given thatdata acquisition is triggered by the fluorescent label associated withthe surface marker for erythroblasts/reticulocytes. Consequently,processing is limited to, e.g., CD71-positive RET, and calculation ofthe frequency of MN-RET in the sample proceed in otherwise standardfashion.

A second aspect of the present invention relates to a method for theenumeration of micronucleated erythrocyte populations. This method canbe carried out by providing a fixed sample comprising erythrocytepopulations including NCE, RET, MN-NCE, MN-RET, or combinations thereof,with the erythrocyte populations being in suspension and substantiallyfree of aggregates, permeable to a nucleic acid dye and RNase, with cellsurface markers in a form recognizable by an antibody, and able toexhibit substantially low autofluorescence; substantially degrading RNAof reticulocytes in the fixed sample with RNase; contacting the fixedsample with a first fluorescent labeled antibody having bindingspecificity for a surface marker for reticulocytes; staining cellularDNA with a nucleic acid staining dye having a fluorescent emissionspectrum which does not substantially overlap with the fluorescentemission spectrum of the first fluorescent labeled antibody; excitingthe nucleic acid staining dye and the fluorescent label associated withthe RET using light of appropriate excitation wavelength for both thenucleic acid staining dye and the fluorescent label to producefluorescent emission; and detecting the fluorescent emission and lightscatter produced by the erythrocyte populations, and counting the numberof RET and MN-RET in said sample while excluding NCE from said counting.

A third aspect of the present invention relates to a method of assessingthe DNA-damaging potential of a chemical agent. This method can becarried out by administering a chemical agent to a mammalian subject andperforming the method according to the first or second aspects of thepresent invention on a peripheral blood or bone marrow sample of themammalian subject, wherein a significant deviation in the percentage ofMN-NCE and/or MN-RET from a baseline MN-NCE and/or MN-RET value inunexposed mammals indicates the genotoxic potential of the chemicalagent. Alternately, each subject may contribute a before treatment bloodor bone marrow specimen. These specimens thus provide subject-specificMN-RET values against which post-treatment MN-RET values can becompared.

A fourth aspect of the present invention relates to a method ofidentifying individuals hypersensitive or insensitive to a DNA-damagingagent. This aspect of the present invention can be carried out byadministering a DNA-damaging agent to a mammalian subject; and thenperforming the method according to the first or second aspects of theinvention on a peripheral blood or bone marrow sample of the mammaliansubject, wherein a significant deviation in the percentage of MN-RETfrom MN-RET values in similarly exposed mammals considered of “normalsensitivity” would indicate the hypersensitivity or insensitivity of themammalian subject to the DNA-damaging agent.

A fifth aspect of the present invention relates to a method of measuringsafety of individuals exposed to one or more suspected DNA-damagingagents in an environment (workplace or other locales of interest). Thisaspect of the present invention can be carried out by performing themethod according to the first or second aspects of the invention usingperipheral blood or bone marrow samples obtained from mammals exposed toone or more DNA-damaging agents in an environment, wherein a significantdeviation in the percentage of MN-RET from a baseline MN-RET value inunexposed mammals indicates that the environment contains one or moreDNA-damaging agents. Alternately, each subject may contribute a beforeexposure blood or bone marrow specimen. These specimens thus providesubject-specific MN-RET values against which post-exposure MN-RET valuescan be compared.

A sixth aspect of the present invention relates to a method ofevaluating the effects of an agent which can modify endogenous orexogenous-induced DNA damage. This aspect of the present invention canbe carried out by administering an agent that may modify endogenous orexogenous-induced genetic damage to a mammalian subject; and thenperforming the method according to the first or second aspects of theinvention on a peripheral blood or bone marrow sample of the mammaliansubject, wherein a significant deviation in the percentage of MN-RETfrom MN-RET values in mammals which are similarly treated except for thesuspected modulating agent indicates that the agent can modifyendogenous or exogenous-induced DNA damage. Alternately, each subjectmay contribute a before treatment blood or bone marrow specimen. Thesespecimens thus provide subject-specific MN-RET values against whichpost-treatment MN-RET values can be compared.

A seventh aspect of the present invention relates to a method ofevaluating the effects of a diet or a dietary nutrient which can modifyendogenous or exogenous-induced DNA damage. This aspect of the presentinvention can be carried out by subjecting a mammal to a predetermineddiet or a dietary nutrient that may modify endogenous orexogenous-induced DNA damage; and then performing the method accordingto the first or second aspects of the invention on a peripheral blood orbone marrow sample of the mammal, wherein (i) a significant deviation inthe percentage of MN-RET from baseline MN-RET values in unexposedmammals indicates that the diet or dietary nutrient can modifyendogenous DNA damage; or (ii) a significant deviation in the percentageof MN-RET from MN-RET values in mammals treated with the samegenotoxicant but without the predetermined diet or the dietary nutrientindicates that the diet or the dietary nutrient can modify exogenous DNAdamage.

An eighth aspect of the present invention relates to a method ofevaluating the effects of a mutation or gene polymorphism which canmodify endogenous or exogenous-induced DNA damage. This aspect of thepresent invention can be carried out by obtaining DNA sequenceinformation for one or more genes of interest for a mammalian subject;and then performing the method according to the first or second aspectof the invention on a peripheral blood or bone marrow sample of themammalian subject, wherein a significant deviation in the percentage ofMN-RET values in mammals with a mutation or gene polymorphism comparedto MN-RET values in similarly treated mammals with a wildtype genotypeindicates that the mutation or gene polymorphism can modify endogenousor exogenous-induced DNA damage.

A ninth aspect of the present invention relates to a method of measuringthe level of DNA damage following exposure of individual(s) to a DNAdamaging agent. This aspect of the present invention can be carried outby performing the method according to the first or second aspects of theinvention on a peripheral blood or bone marrow sample of a mammalexposed to a DNA damaging agent, wherein a significant deviation in thepercentage of MN-RET from a baseline MN-RET value in unexposed mammalsindicates that the agent caused DNA damage and wherein greater deviationfrom the normal percentage indicates the level of the DNA damage.Alternately, each subject may contribute a before treatment blood orbone marrow specimen. These specimens thus provide subject-specificMN-RET values against which post-treatment MN-RET values can becompared.

A tenth aspect of the present invention relates to a method of assessingasplenia or hyposplenic function. This aspect of the present inventioncan be carried out by performing the method according to the first orsecond aspects of the invention on a peripheral blood sample of amammal, wherein either (i) a significant deviation in the percentage ofMN-NCE from a baseline MN-NCE value in normal mammals possessing ahealthy functional spleen, (ii) a ratio of MN-RET frequency to MN-NCEfrequency is less than about 20, or (iii) both (i) and (ii), indicatesasplenia or hyposplenic function. This aspect of the present inventioncan be used to assess splenic dysfunction that is associated with adisease state, or which results from exposure to toxic agent(s).

An eleventh aspect of the present invention relates to a method ofassessing the efficacy of drugs or other interventions such as dietarychanges for preventing or delaying the onset of asplenia or hyposplenicfunction. This aspect of the present invention can be carried out byperforming the method according to the first or second aspects of theinvention on a peripheral blood sample of a mammal, wherein the changein MN-NCE frequency over time and/or the ratio of MN-RET frequency toMN-NCE frequency is compared to a historical database which describesthe typical rate at which these values increase for subjects with thesame disease or condition associated with asplenia or hyposplenicfunction. Efficacy would then be indicated by a lower rate of change toMN-NCE frequency, and/or the MN-RET to MN-NCE ratio. Alternately,efficacy could be determined by grouping subjects with similar diseasesor conditions known to result in asplenia or hyposplenic function intotreatment and placebo groups. Efficacy would be exhibited by a lowerrate of MN-NCE increase, and/or a lower rate of increase to the ratioMN-RET to MN-NCE for those subjects undergoing treatment with thepresumptive protecting agent.

A twelfth aspect of the present invention relates to a method wherebyerythrophagocytic activity measurements provide prognostic informationregarding the likely severity of diseases or conditions associated withhyposplenism or functional asplenia. This aspect of the presentinvention can be carried out by performing the method according to thefirst or second aspects of the invention on a peripheral blood sample ofa mammal, wherein the change in MN-NCE frequency over time and/or theratio of MN-RET frequency to MN-NCE frequency is compared to ahistorical database which describes the typical rate of change forsubjects with the same disease or condition associated with asplenia orhyposplenic function. Significant departures from this average rate ofchange likely reflects accumulated damage to the spleen, which in turnmay be representative of global organ damage. Subjects that haveelevated MN-NCE and/or MN-RET to MN-NCE ratios early in life, or whichchange substantially over a short period of time may be predicted tohave a more severe form of the disease, whereby more vigorousinterventions may be indicated. Conversely, subjects whose MN-NCE and/orMN-RET to MN-NCE ratios rise appreciably more slowly than usual forthese diseases and conditions may be less at risk for complications andtherefore less aggressive monitoring and/or intervention may bedesirable.

A thirteenth aspect of the present invention relates to a method ofassessing anemia. This aspect of the present invention can be carriedout by performing the method according to the first or second aspects ofthe invention on a peripheral blood or bone marrow sample of a mammal,wherein a significant deviation in the percentage of MN-RET from abaseline MN-RET value in normal mammals assists the differentialdiagnosis of anemia.

A fourteenth aspect of the present invention relates to a method ofassessing severity of a disease or disorder associated with hyposplenicfunction. This aspect of the present invention can be carried out byperforming the method according to the first or second aspect of theinvention on a peripheral blood sample of a human having a disease ordisorder associated with hyposplenic function; and then determining aratio of micronucleated reticulocyte frequency to micronucleatednormochromatic erythrocyte frequency, wherein severity of the disease ordisorder is indicated by the smaller the ratio is when less than about20.

A fifteenth aspect of the present invention relates to a kit thatincludes one or more reagents for practicing the various aspects of thepresent invention. The kit preferably includes, a first containerholding a solution that includes a first antibody that recognizes a cellsurface marker for reticulocytes; a second container holding a solutionthat includes a second antibody that recognizes a cell surface markerfor platelets; and a third container holding a nucleic acid dye.

The labeling/staining procedure described in the current report preventsplatelets and platelet-associated aggregates from affecting MNmeasurements, and is based on a nucleic acid dye with higher specificityfor DNA. Significantly, the scoring system described herein quantifiesMN frequency in the most immature fraction of RET, and accomplishes thisat previously unattainable rates of speed. Beyond describing an improvedmethodological approach for enumerating MN-RET, this report includesdata from experiments which indicates that exposure to knownDNA-damaging agents induce MN-RET which can be detected in peripheralblood circulation of eusplenic humans. Beyond describing an improvedmethodological approach for enumerating MN-NCE, this report includesdata from experiments which indicates that spleen dysfunction stemmingfrom vaso-occlusive events results in elevated MN-NCE frequencies whichcan be detected in peripheral blood circulation of humans.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bivariate graph of malaria-infected rat blood. Greenfluorescence associated with CD71 expression is graphed on the y-axis,and red fluorescence associated with DNA content is graphed on thex-axis. Note that nucleated cells, which fall in the fourth decade ofpropidium iodide fluorescence, have been excluded from this plot basedon their high (2n) DNA content. Malaria-infected blood was stained inparallel with test samples and analyzed at the beginning of each day ofanalysis. These samples were used to set appropriate PMT voltages andelectronic compensation. As malaria-infected erythrocytes mimic the DNAcontent of micronucleated erythrocytes, they also served to guide theposition of the quadrant that was used to distinguish erythrocytes withand without micronuclei.

FIGS. 2A-B illustrate flow cytometry-based MN-RET frequencies graphedfor blood samples obtained from five rats on six consecutive days. (Notethat blood samples for Days 0-4 were collected from the tail vein, andDay 5 were collected via heart puncture). For comparison purposes,corresponding microscopy-based values for Day 5 are shown to the farright. When applied to tail vein specimens, the 2-color method (FIG. 2A)is seen to provide MN-RET values which are considerably more variablethan those associated with 3-color analyses (FIG. 2B). Conversely,measurements associated with heart blood specimens were in goodagreement among microscopy, and 2- and 3-color flow cytometrytechniques.

FIGS. 3A-C are bivariate graphs that illustrate the gating strategy usedfor mammalian blood analyses based on the 3-color labeling proceduredescribed herein. In order to be evaluated for micronuclei, cells mustfall within a light scatter region which corresponds to singleunaggregated cells (FIG. 3A), exhibit a sub-2n DNA content (FIG. 3B),and lack expression of CD42b (a platelet-specific antigen) (FIG. 3C).

FIGS. 4A-B are bivariate graphs of blood samples from cancer patientch1, with FIG. 4A showing a pre-treatment sample and FIG. 4B showing asample collected 72 h after treatment with 60 mg cisplatin and 100 mgetoposide per m². CD71-associated fluorescence is graphed on the y-axis,and propidium iodide-associated with DNA content is graphed on thex-axis. Approximately 1.5 million events are shown in each bivariateplot. Comparison of FIGS. 4A-B show a reduction in reticulocytes (RET,upper left quadrant) and an increased frequency of micronucleatedreticulocytes (MN-RET, upper right quadrant) in the 72 h post-treatmentsample.

FIG. 5 is a bivariate graph illustrating the resolution of propidiumiodide-positive erythrocytes (i.e., RNA positive RET) andanti-CD71-FITC-positive erythrocytes (i.e., very immature RET) fromnormochromatic erythrocytes. For this analysis, fixed samples weretreated with the standard reagents as described; however, RNase wasomitted. These analyses suggest that it is approximately the youngest10% of RNA-positive human RET in peripheral blood circulation which arelabeled with the anti-CD71 reagent.

FIG. 6 illustrates CD71-positive micronucleated reticulocytes(MN-RET^(CD71+) (%)) and CD71-positive reticulocytes (RET^(CD71+) (%))which are graphed for nine cancer therapy patient (rt: radiotherapy; ch:chemotherapy). While the frequency of RET^(CD71+) was generally found todecline over the first week of treatment, higher incidences ofMN-RET^(CD71+) were observed. The time-dependent increase inMN-RET^(CD71+) (%) for chemo- and radiotherapy cancer patients arestatistically significant (P=0.0166 and 0.0081, respectively).

FIG. 7 illustrates a model describing the major factors that affectmicronucleated reticulocyte (MN-RET) frequency in human peripheralblood. (A) Radiation intensity or chemical dose; (B) host-specificfactors which dictate intrinsic chemo- or radiosensitivity; (C) dilutioneffect as induced MN-RET enter peripheral blood circulation withreticulocytes derived from unexposed red marrow site(s); (D)erythrophagocytosis activity (especially the spleen) removesMN-containing red blood cells from circulation.

FIG. 8 is a graph illustrating average flow cytometry-basedmicronucleated reticulocyte frequencies (with S.E.M. bars) for humanblood samples obtained from a chemotherapy patient before and aftertreatment. These specimens were analyzed in triplicate using the 2-colorand 3-color labeling procedures (left and center bars). Additionally,triplicate samples were analyzed at very high density using the 3-colorprocedure in conjunction with FL1 thresholding (right bars). The 2-colorspecimens exhibited spurious events in the MN-RET quadrant, andconsequently these values tended to be higher than their 3-colorcounterparts. The high density/FL1 thresholding technique was observedto reduce data acquisition time and the size of FCM files significantly.

FIGS. 9A-B illustrate the frequency of MN-NCE versus patient age.(MN-NCE are labeled as the number of mature red blood cells withHowell-Jolly bodies per million cells.) FIG. 9A corresponds to sicklecell anemia patients with the most severe form of the disease, HbSS.FIG. 9B corresponds to sickle cell anemia patients with the generallymild form of the disease, HbSC. The significant age-dependent increasein MN-NCE values observed for young HbSS patients is likely related tothe degree to which accumulated vaso-occlusive damage has destroyedsplenic architecture and filtration function. Regarding HbSC disease,only two of twelve patients exhibited MN-NCE values that are suggestiveof splenic dysfunction.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method for the enumeration ofmicronucleated erythrocyte populations using an optical device designedfor illumination and analysis of blood samples.

For purposes of the present invention, “erythrocyte populations” isintended to include, among other blood cells, populations of NCE, RET,MN-NCE, MN-RET, and combinations thereof. Samples of erythrocytepopulations from mammals can be obtained from either peripheral blood orbone marrow. The erythrocyte populations from any mammal can be analyzedin accordance with the present invention, although preferred mammalsinclude, without limitation, rodents, such as rat and mouse; canines,such as beagle dogs; and primates such as monkeys, chimpanzees, andhumans. As for the source of mammalian erythrocytes, conventionalprocedures can be utilized to obtain samples. For example, a bloodsample can be obtained from the tail vein of rodents after a briefwarming period under a heat lamp. Alternately, cardiac puncture may beperformed on anesthetized animals. In the case of humans, a finger prickwith a lancet or a blood draw via standard venipuncture are convenientsources of erythrocytes. In any case, blood should be collected into ananticoagulant (e.g., EDTA or heparin) to prevent aggregation and clotformation. Bone marrow samples can also be acquired according tostandard procedures. Standard buffers which do not lead to cellularaggregation or clotting should be utilized with bone marrow samples. Thesamples can also be treated in a manner that affords enrichment of theerythrocyte populations to be examined (Abramsson-Zetterberg et al.,“Human Cytogenetic Biomonitoring Using Flow-Cytometric Analysis ofMicronuclei in Transferrin-Positive Immature Peripheral BloodReticulocytes,” Environ. Mol. Mutagen. 36:22-31 (2000); Choy andMacGregor, “Density-gradient Enrichment of Newly-Formed MouseErythrocytes: Application to the Micronucleus Test,” Mutat. Res.130:159-164 (1984), which is hereby incorporated by reference in itsentirety), although enrichment is a less preferred approach given thatadditional steps are required and the enrichment process may skew theresults of any frequency analysis.

Even when sample collection occurs in a manner designed to reduce thelikelihood of gross clot formation, some degree of platelet-platelet andplatelet-cell aggregation often occurs (hereafter referred to as“platelet-associated aggregates”). These events have the potential tointerfere with the accurate enumeration of red blood cell populations,including MN-containing erythrocytes. It would be desirable, therefore,to provide a means for discriminating platelet-associated aggregates,and even singular platelets, from red blood cells of interest (i.e.,NCE, RET, MN-NCE, and MN-RET). A method which labels platelets, but notred blood cells, with a fluorochrome-conjugated antibody is anadvantageous means for accomplishing this, as it eliminates the need forprocessing steps designed to physically separate red blood cells fromplatelets.

Once a blood or bone marrow sample has been obtained, the sample isfixed so as to render the blood cells in suspension and preferablysubstantially (but not necessarily completely) free of aggregates,permeable to a nucleic acid dye and RNase, with cell surface markersintact (i.e., in a form recognizable by appropriate antibodies), andexhibiting substantially low autofluorescence. Fixing is accomplished inalcohol at a temperature of about −40° C. to about −90° C. Briefly, a100 to 1000 μl aliquot of each blood suspension (e.g., from a syringeand needle or from a pipettor) is delivered forcefully into tubescontaining a suitable amount (e.g., about 1 to about 11 ml) of ultracoldalcohol. It is preferable that the ultracold alcohol fixative ismaintained at about −40° C. to about −90° C., preferably about −70° C.to about −90° C. The alcohol may be a primary alcohol or a secondaryalcohol. Suitable primary alcohols include but are not limited toethanol and methanol. Suitable secondary alcohols include but are notlimited to isopropyl alcohol. Of these alcohols, methanol is preferred.Once the samples are fixed, the tubes can be struck sharply or vortexedto break up aggregates. The samples can be stored at about −40° C. toabout −90° C., preferably about −70° C. to about −90° C. The samples arepreferably stored overnight (e.g., between about 8-15 hours) prior toanalysis.

Prior to analysis, the cells are diluted out of the fixative with icecold buffered salt solution. In a preferred embodiment, the bufferedsalt solution is Hank's Balanced Salt Solution (HBSS), or about 0.9%NaCl supplemented with sodium bicarbonate, preferably at about 5.3 mM.The cells are centrifuged under conditions which are effective atmaintaining cell structure while removing dissolved solids therefrom.Exemplary centrifugation conditions include about 500× to about 1000×gfor about 5 minutes. Thereafter, supernatants are decanted and the cellpellets can be stored at about 4° C. or on ice until analysis. Oncecells are washed out of alcohol fixative, it is preferable to stain andanalyze them within about 3 days, more preferably on the same day thatthey are washed out of fixative.

Once the cells are washed out of fixative, RNA of the reticulocytes issubstantially degraded with RNase so that the only nucleic acid thatremains is DNA (i.e., DNA of micronuclei or Howell-Jolly bodies, ifpresent). RNase treatment can be carried out by introducing fixed andwashed erythrocyte populations into tubes containing an appropriateamount of an RNase A solution (i.e., ˜20 μg RNase/ml HBSS). Incubationswith RNase are preferably carried out at about 4° C. to about 25° C.

Following RNase treatment, nucleic acid dyes are used to stain DNA ofmicronuclei present in erythrocytes or reticulocytes and fluorescentlabeled antibodies directed to specific cell surface markers are used todistinguish reticulocytes from more mature erythrocytes, platelets, andplatelet-associated aggregates, as well as to distinguish onesub-population from another sub-population within the larger erythrocytepopulation. Alternatively, RNase treatment and antibody marking ofreticulocytes and platelets can be carried out simultaneously.

One type of antibody employed in the present invention has bindingspecificity for a surface marker for reticulocytes and includes afluorescent label with a fluorescent emission pattern that is detectableby the optical detection equipment employed. As used herein, “a surfacemarker for erythroblasts/reticulocytes” means at least one species of asurface antigen present on reticulocytes but absent on matureerythrocytes, thereby enabling reticulocytes (and erythroblasts) to bedistinguished from mature erythrocytes by the presence of this marker.Such markers are known in the art to include, but are not limited to,CD71 (a transferrin receptor). It should be appreciated by those ofordinary skill in the art that other reticulocyte cell surface markershave been and may continue to be identified, and antibodies directed tosuch markers can likewise be employed.

Another type of antibody employed in the present invention has bindingspecificity for a surface marker for platelets and includes afluorescent label with a fluorescent emission pattern that is detectableby the optical detection equipment employed. As used herein, “a surfacemarker for platelets” means at least one species of a surface antigenpresent on platelets but absent on reticulocytes and erythrocytes,thereby enabling platelets to be distinguished by the presence of thismarker. Such markers are known in the art to include, but are notlimited to, CD9, CDw17, CD29, CD31, CD32, CD41, CD42a, CD42b, CD42c,CD42d, CD43, CD46, CD49f, CD51, CD60a, CD61, CD62P, CD63, CD69, CD82,CD98, CD102, CD110, CD112, CDw119, CD120a, CD128a, CD128b, CD130, CD132,CD140a, CD141, CD148, CD151, CD165, CD184, CD226, and CD245. It shouldbe appreciated by those of ordinary skill in the art that other plateletsurface markers have been and may continue to be identified, andantibodies directed to such markers can likewise be employed.

In addition to the use of whole antibodies (e.g., polyclonal ormonoclonal antibodies), it should be appreciated that whole antibodiescan be substituted by using binding portions of such antibodies. Suchbinding portions include, without limitation, Fab fragments, F(ab′)₂fragments, and Fv fragments. As used herein, Fab fragments, F(ab′)₂fragments, and Fv fragments are functional equivalents of wholeantibodies.

A number of fluorescent labels are available which have the desiredexcitation and emission characteristics. As used herein, the term“fluorescent label” means at least one species of a fluorescent moleculethat is conjugated or otherwise attached to a monoclonal antibody withbinding specificity for a surface marker for erythroblasts/reticulocytesor a surface marker for platelets. Because optical detection equipmentis intended to be employed with the present invention, the selectedfluorescent label used on the antibodies should accommodate theexcitation parameters of the illuminating light source employed in theoptical detection equipment.

Where multiple antibodies are used to label different erythrocytesub-populations and even other events such as platelets andplatelet-associated aggregates, then it is desirable to utilizedifferent fluorescent labels on each type of antibody, such that eachlabel has an emission spectrum which does not substantially overlap theemission spectra of other labels. Preferably, each has a sufficientlydistinct emission maxima that discriminates itself from otherfluorescent labels.

Generally, fluorescent labels having an excitation wavelength that ismatched to the wavelength of illuminating light, which is typically inthe range of about 485 to about 491 nm. The fluorescent labels can haveany that can be detected by an appropriate detector device. By way ofexample, many fluorescent labels have an emission maxima in a range ofabout 510 to about 750 nm. Suitable fluorescent labels include, but arenot limited to fluorescein isothiocyanate (FITC), Alexa Fluor 488,phycoerytherin (PE), PE-Texas Red, PE-Cy5, PerCP, PerCP-Cy5.5, andPE-Cy7.

A preferred fluorescent labeled antibody directed to a surface markerfor erythroblasts/reticulocytes (i.e., discriminating between RET andmature erythrocytes) is anti-CD71-FITC antibody.

A preferred fluorescent labeled antibody directed to human platelets orplatelet-associated aggregates is anti-CD42b-PE. A preferred fluorescentlabeled antibody directed to rodent platelets or platelet-associatedaggregates is anti-CD61-PE.

Labeling of erythroblast/reticulocyte and/or platelets andplatelet-associated aggregates with selected fluorescent labeledantibodies is achieved by combining antibody solution with the fixed andwashed mammalian blood (or bone marrow) sample under conditionseffective to allow antibodies to recognize the cell or platelet surfacemarkers. Exemplary conditions include an approximately 30 minuteincubation period at about 4° C. Thereafter, sample can be washed using,e.g. buffered saline solution or HBSS (with or without fetal bovineserum, at about 1% volume/volume).

Suitable nucleic acid dyes are those capable of staining cellular DNA ata concentration range detectable by the optical detection equipment andwhich have a fluorescent emission spectrum which does not substantially(i.e., significantly) overlap with the fluorescent emission spectrum ofthe fluorescent labels used on antibodies. A preferred nucleic acid dyeis propidium iodide. It should be appreciated by those of ordinary skillin the art that other nucleic acid dyes are known in the art and arecontinually being identified. Any suitable nucleic acid dye withappropriate excitation and emission patterns can be utilized herein.

Washed antibody-labeled cells can be resuspended with a nucleic acid dyesolution (e.g., dilution of dye stock solution in HBSS). Nucleic aciddyes are available from a number of suppliers in crystalline form or ashighly concentrated stock solutions. It is desirable to work withnucleic acid dyes once cell density and dye concentration parametershave been optimized through routine experimentation as described in theExamples infra.

Thereafter, the treated sample can be subjected to optical detection ofthe micronucleated erythrocyte populations.

The optical detection systems have one or more light sources, preferablyin the form of one or more amplified or collimated beams of light, thatare able to excite the nucleic acid dye(s) and fluorescent labeledantibodies; and one or more detectors that are able to detect thefluorescence emissions caused by the nucleic acid dye(s) and thefluorescent labeled antibodies. Suitable optical detection systemsinclude, without limitation, single-laser flow cytometers; dual- ormultiple-laser flow cytometers; and hematology analyzers equipped withan appropriate illumination device (e.g., diode, laser, etc.).

Single-laser flow cytometric analysis uses a single focused laser beamwith an appropriate emission band to excite the nucleic acid dye(s) andthe fluorescent labeled antibodies. As cells pass through the focusedlaser beam, the cells bound by anti-reticulocyte/erythroblast antibodyexhibit a fluorescent emission maxima characteristic of the fluorescentlabel associated therewith, cells possessing a micronucleus exhibit afluorescent emission maxima characteristic of the nucleic acid dye, andevents (i.e., platelets or platelet-associated aggregates) bound byanti-platelet antibody exhibit a fluorescent emission maximacharacteristic of the fluorescent label associated therewith. The flowcytometer is equipped with appropriate detection devices to enabledetection of the fluorescent emissions and light scatter produced by theerythrocyte populations and the platelets. Cells are counted and thenumber of specific erythrocyte sub-populations in the sample can becounted and, importantly, discriminated from platelets andplatelet-associated aggregates.

Dual- or multiple-laser flow cytometric analysis use two or more focusedlaser beams with appropriate emission bands, in much the same manner asdescribed above for the single-laser flow cytometer. Different emissionbands afforded by the two or more lasers allow for additionalcombinations of nucleic acid dye(s) and fluorescent labeled antibodies.

Prior to such excitation and detection of fluorescence from the treatedsamples, the optical detection system can be calibrated for thedetection of micronuclei. This can be achieved using a biologicalstandard which has been treated in parallel with the fixed sample (i.e.,RNase, antibody treatment, nucleic acid stain, etc.). Preferredbiological standards are fixed erythrocyte samples obtained from amalaria-infected mammal, more preferably a Plasmodium berghei-infectedrodent (e.g., rat or mouse). The use of the biological standard mimicsthe micronucleated erythrocytes. As a result of the use of suchbiological standards to calibrate a flow cytometer, for example, it ispossible to achieve one or more of the following: settingphotomultiplier tube voltage, setting electronic compensationparameters, and defining the position of regions that indicatemicronucleus-containing erythrocytes.

According to a modified approach for using high density cell samples, itis possible to utilize the presence of the surface marker forerythroblasts/reticulocytes as a means for excluding cells (e.g., matureerythrocytes) to be counted. Because mature erythrocytes make up thepredominant cell sub-population, it is possible to exclude those cellsfrom counting and thereby improve the speed of collecting data on aparticular sample. For example, it becomes possible to screen sampleshaving densities greater than about 30 million cells/ml, more preferablygreater than about 50 million cells/ml, most preferably greater than 80million cells/ml. As a consequence, the time for counting cells in asample can be significantly reduced. This approach can be carried outeither with or without the use of an antibody that recognizes a surfacemarker for platelets.

The present inventions will find myriad uses in the field of toxicity,and in particular, the field of genetic toxicology. For example, thepresent inventions will be useful for studying (i) whether chemical orphysical agents damage DNA, (ii) whether chemical or physical agentsprotect against endogenous or exogenous DNA damage, (iii) whetherchemical or physical agents potentiate endogenous or exogenous DNAdamage, (iv) whether mutations and/or genetic polymorphisms lead toincreased endogenous or exogenous DNA damage, and (v) whether mutationsand/or genetic polymorphisms lead to decreased endogenous or exogenousDNA damage. In using the methods of the present inventions to assessendogenous or exogenous DNA damage, or for evaluating the influence ofmodulating agents or genotypes on endogenous or exogenous DNA damage,determinations of such effects are typically based on statisticallysignificant differences as measured using appropriate statisticalanalyses.

General experimental design considerations for evaluating DNA damagingagents according to the present inventions would involve administeringthe agent to a mammal prior to obtaining one or more blood samples. Theadministering of the DNA-damaging agent can be performed anywhere fromabout 1 to about 4 days, preferably about 2 to 4 days, prior toobtaining the blood sample. Additionally, one or more pre-exposure bloodsample may be obtained, and would serve as a subject-specific controlfor evaluating treatment related changes to MN-RET frequency. To monitorthe modulating effects of a suspected modulating agent, the suspectedmodulating agent can be administered to the individual simultaneous orcontemporaneous with administration of the DNA-damaging agent. Bycontemporaneous, administration of the modulating agent is intended tooccur before, after, or both before and after administration or exposureto the DNA damaging agent. Preferably, contemporaneous administrationoccurs within about 12 hours (i.e., before and/or after). Any modulatingeffect afforded by the agent can be measured relative to damage causedin the absence of the suspected modulating agent or to historical databased on the degree of damage normally afforded by the DNA-damagingagent.

Therefore, the present inventions can be used to assess the DNA-damagingpotential of a chemical agent (e.g., a pharmaceutical agent) byadministering the chemical agent to a mammalian subject and thenperforming the analysis of the present inventions on a sample from amammalian subject, wherein a significant deviation in the percentage ofMN-RET from a baseline MN-RET value in an unexposed subject (i.e.,placebo-receiving mammalian subject) indicates the genotoxic potentialof the chemical agent. The greater the deviation from the baselinevalue, the greater the extent or level of damage caused by the chemicalagent. Alternately, each subject may contribute one or morebefore-treatment blood or bone marrow specimen. These specimens thusprovide subject-specific MN-RET values against which post-treatmentMN-RET values can be compared. Examples of chemicals that damage DNAinclude, but are not limited to: inorganic genotoxicants (e.g., arsenic,cadmium and nickel), organic genotoxicants (especially those used asantineoplastic drugs, e.g., cyclophosphamide, cisplatin, vinblastine,cytosine arabinoside, etc.), anti-metabolites (especially those used asantineoplastic drugs, e.g., methotrexate and 5-fluorouracil), organicgenotoxicants that are generated by combustion processes (e.g.,polycyclic aromatic hydrocarbons such as benzo(a)pyrene), as well asorganic genotoxicants that are found in nature (e.g., aflatoxins such asaflatoxin B1).

Likewise, the present inventions can be used to assess the DNA-damagingpotential of a physical agent (e.g., ionizing radiation) byadministering the physical agent to a mammalian subject and thenperforming the analysis of the present inventions on a sample from amammalian subject, wherein a significant deviation in the percentage ofMN-RET from a baseline MN-RET value in an unexposed subject (i.e.,sham-exposed mammalian subject) indicates the genotoxic potential of thephysical agent. The greater the deviation from the baseline value, thegreater the extent or level of damage caused by the physical agent.Alternately, each subject may contribute one or more before-treatmentblood or bone marrow specimen. These specimens thus providesubject-specific MN-RET values against which post-treatment MN-RETvalues can be compared. Examples of physical agents known to cause DNAdamage include, but are not limited to: gamma radiation, beta radiation,and UV radiation.

Such monitoring can be used to identify individuals who arehypersensitive or refractory to endogenous or exogenous DNA-damage.Analyses performed according to the present inventions would beconducted on a sample from a mammalian subject, wherein a significantdeviation in the percentage of MN-RET from MN-RET values in similarlytreated mammals considered of “normal sensitivity” would indicate thedegree of hypersensitivity or insensitivity. When DNA sequence data areavailable and combined with MN-RET measurements provided by the presentinventions, then it becomes possible to identify mutations and/orgenetic polymorphisms that convey hypersensitivity or insensitivityphenotypes for endogenous or exogenous DNA-damage.

Furthermore, as part of a routine protocol following an adverse event ina particular environment (e.g., radiation leak or carcinogenic agentspill), such monitoring can be used to define the extent of harm anenvironment presents as well as the successfulness of any cleanup. Theaffected environment is typically, but not necessarily limited to, aworkplace environment. These monitoring approaches can be carried out byperforming the analysis of the present inventions using samples obtainedfrom mammals exposed to one or more DNA-damaging agents in the affectedenvironment, wherein a significant deviation in the percentage of MN-RETfrom a baseline MN-RET value in unexposed mammals indicates that theaffected environment contains one or more DNA-damaging agents. Inaddition, the level of damage caused by such agents to which the mammalswere exposed indicates the severity of contamination to the affectedenvironment. Alternately, each subject may contribute one or morebefore-exposure blood or bone marrow specimen. These specimens thusprovide subject-specific MN-RET values against which post-exposureMN-RET values can be compared.

Because of the interaction of agents, it is possible that certain agentsmay offer protective benefit while other agents may present a magnifiedrisk when combined. For this reason, the present inventions can be usedto evaluate the effects of an agent which can modify (i.e., enhance orsuppress) endogenous or exogenous-induced DNA damage. This can beachieved by subjecting mammals to a suspected modulating agent with orwithout exposure to an exogenous agent that can induce DNA damage andthen performing the analysis of the present inventions on a sample fromthe subject. A significant deviation in the percentage of MN-RET frombaseline MN-RET values in unexposed mammals indicates that the agent canmodify endogenous DNA damage; a significant deviation in the percentageof MN-RET from MN-RET values in mammals treated with the same exogenousgenotoxicant but without the modifying agent indicates that the agentcan modify exogenous DNA damage. A reduction in the percentage of MN-RETcompared to baseline figures indicates a suppression of DNA-induceddamage, whereas an increase in the percentage of MN-RET indicates anenhancement of DNA-induced damage.

Putative protective agents can be vitamins, bioflavonoids andanti-oxidants, dietary supplements (e.g., herbal supplements), anddietary adjustments (e.g., diets high in beneficial foods and low inprocessed foods), or any other protective agent, naturally occurring orsynthesized by man.

As noted above, diet and dietary nutrients are one type of potentiallyprotective agents. Thus, another aspect of the invention relates to amethod of evaluating the effects of a diet or a dietary nutrient whichcan modify endogenous or exogenous-induced DNA damage. This can beachieved by subjecting a mammal to a predetermined diet or a dietarynutrient that may modify endogenous or exogenous-induced DNA damage,either with or without exposure to exogenous agents that can induced DNAdamage. The analysis of the present inventions is performed on samplesfrom the mammal, wherein a significant deviation in the percentage ofMN-RET from a baseline MN-RET value in unexposed mammals indicates thatthe diet can modify endogenous DNA damage. A significant deviation inthe percentage of MN-RET from MN-RET values in mammals treated with thesame exogenous genotoxicant but without the diet or dietary nutrientindicates that the diet or dietary nutrient can modify exogenous DNAdamage.

Further aspects of the present inventions relate to its use fordiagnosis and the monitoring of certain diseases. Whereas in the fieldof toxicology and genetic toxicology red blood cell inclusions formed ofchromatin are known as micronuclei, the field of medical hematology hasknown them as Howell-Jolly bodies (“HJB”). In the medical hematologyfield, when HJB are observed upon microscopic inspection of peripheralblood smears, they are considered evidence of dysfunctional (or missing)spleen, or of certain disease states. For instance, HJB are observed athigh frequencies in patients with Megaloblastic anemia. Thus,differentiating this type of anemia from others can in part be aided byan assessment of HJB frequency. For instance, if Pernicious anemia (atype of Megaloblastic anemia) is suspected, then B12 administration isindicated. One way of assessing whether the diagnosis was correct andwhether treatment has been effective would be to use the presentinventions to evaluate the frequency of MN-RET and/or MN-NCE before andover the course of therapy.

Another use of the present inventions in the area of clinical diagnosisor patient monitoring is assessment of spleen function. The human spleenis primarily responsible for eliminating HJB-containing red blood cells.In fact, the healthy human spleen is able to reduce HJB-containingerythrocytes from an average of about 0.1% to about 0.3% in the bonemarrow, and to about 0.002% in peripheral blood circulation. Thus, whenthe frequency of HJB-containing normochromatic erythrocytes (MN-NCE)increases, this is indicative of splenic dysfunction. There aredisease-related states that can result in compromised splenic function.These conditions are important to detect, since subjects with aspleniaor hyposplenic function can be at increased risk of infection byencapsulated organisms such as pneumococci, Haemophilus influenzae, andmeningococci. These patients are also more susceptible to infectionswith intra-erythrocytic organisms such as Babesia microti and those thatseldom affect healthy people, such as Capnocytophaga canimorsus. This iswhy, for instance, patients with sickle cell disease are oftenprophylactically treated with antibiotics to compensate for the spleendysfunction which occurs as a secondary result of their disease. Otherdisease states that are associated with functional asplenia orhyposplenic function include: Celiac disease, cirrhosis with or withoutportal hypertension, vasculitis, systemic lupus erythematosus or discoidlupus. Bone marrow transplantation can also result in hyposplenicfunction. The present inventions could therefore detect the presence ofasplenia or hyposplenic function, as increased incidence of peripheralblood MN-NCE reflects absent or impaired spleen erythrophagocytosisactivity.

A further aspect of the present inventions regards its potential in thefield of medicine, whereby the severity of diseases associated withsplenic dysfunction can be predicted based on MN-RET and/or MN-NCEmeasurements. The analysis of the present inventions can be performed onone or more samples from the subject, wherein the MN-NCE frequencyand/or the ratio of MN-RET frequency to MN-NCE frequency is compared toeither (i) a historical database that describes the typical value forsubjects with the same disease or condition associated with asplenia orhyposplenic function, or (ii) based on prior measurements performed onsamples obtained from the patient. Subjects that have elevated MN-NCEand/or MN-RET to MN-NCE ratios early in life, or which changesubstantially over a short period of time may be predicted to have amore severe form of the disease, whereby more vigorous interventions maybe indicated. Conversely, subjects whose MN-NCE and/or MN-RET to MN-NCEratios rise appreciably more slowly than usual for these diseases andconditions may be less at risk for complications and therefore lessaggressive monitoring and/or intervention may be desirable.

Also contemplated is a kit to facilitate practice of the presentinvention. The kit can include any one or more of the above-identifiedreagents (present in multiple containers), materials (e.g., sampletubes), and optionally an instruction manual. A preferred kit of thepresent invention will contain at least one antibody that recognizes acell surface marker for reticulocytes, at least one antibody thatrecognizes a surface marker for platelets, and a nucleic acid dye. Morepreferably, the kit can further contain a fixative agent, a bufferedsalt solutions, RNase, a biological standard, and suitable tubes for thecollection and/or centrifugation of samples. The kit can optionallyinclude software templates the identify parameters of operation anddetection for cellular events.

EXAMPLES

The examples below are intended to exemplify the practice of the presentinvention but are by no means intended to limit the scope thereof.

Example 1 Rodent Blood Specimens

Sprague-Dawley rats (4-5 weeks old) were purchased from Charles RiverLaboratories. Animals were housed two per cage and assigned randomly totreatment groups. The animals were acclimated for approximately 2 weeksbefore experiments were initiated, with food and water available adlibitum throughout the acclimation and experimentation periods. Ratswere treated via intraperitoneal injection with 0.9% saline for fivedays. Before each of the daily treatments, “low volume” blood specimens(approximately 100 μl) were collected from the tail vein of each animalinto heparinized Phosphate Buffered Saline solution (i.e., blood wasdrawn into anticoagulant-filled 26.5 gauge needles and syringes after abrief warming period under a heat lamp). On the terminal blood harvestday, “high volume” blood samples were collected. The high volume bloodcollection occurred via heart puncture into anticoagulant-filled needleand syringe (approximately 1.2 blood to 5 ml anticoagulant solution).Low and high volume blood specimens were fixed for flow cytometricenumeration of MN-RET frequencies according to methods of the presentinvention. Fixed, coded blood specimens were stored at −80° C. untilflow cytometric analysis. Heart puncture blood specimens were also addedto an equal volume of heat-inactivated fetal bovine serum and smearedonto clean microscope slides, allowed to air dry, and then fixed withabsolute methanol for ten minutes. Coded slides were stored in a slidebox until they were processed for microscopy-based MN-RET scoringaccording to standard practices.

For flow cytometric analysis, rat blood samples were washed out offixative with HBSS, and were stained according to “2-color” and“3-color” labeling methods. For the 2-color method, 20 μl of fixed,washed cells were added to flow cytometry tubes containing 80 μl of anRNase/antibody solution (contains 10 μl anti-rat CD71-FITC antibody and10 μg RNase A per ml HBSS). Following successive 30 minute incubationsat 4° C. and room temperature, cells were resuspended in 1-2 mlpropidium iodide solution (1.25 μg/ml HBSS). Tubes were stored at 4° C.until analysis (same day). For 3-color analyses, the same reagents andincubation times were utilized, with the exception that anti-CD61-PE wasincluded in the RNase/antibody solution at 5 μl per ml.

At the beginning of each day of flow cytometric analysis,instrumentation and acquisition/analysis software parameters werecalibrated based on the fluorescence of a biological standard:malaria-infected rat blood (Dertinger et al., “Malaria-infectedErythrocytes Serve as Biological Standards to Ensure Reliable andConsistent Scoring of Micronucleated Erythrocytes by Flow Cytometry,”Mutat. Res. 464:195-200 (2000), which is hereby incorporated byreference in its entirety). This sample guided PMT voltage settings tooptimally resolve parasitized (MN-like) reticulocytes, and the positionof the quadrant which delineated erythrocytes with and without MN. Thehigh prevalence of reticulocytes (i.e., FITC-positive events) andmalaria-infected reticulocytes (i.e., FITC- and propidiumiodide-positive events) also helped guide compensation settings. SeeFIG. 1.

CELLQuest software v3.3 (BD-Immunocytometry Systems, San Jose, Calif.),was utilized for data acquisition and analysis. Events were triggered onthe forward scatter parameter. Data collection for each sample proceededuntil the number of CD71-positive RET (RET^(CD71+)) equaled 20,000. Thefrequency of micronucleus-containing CD71-positive reticulocytes(MN-RET^(CD71+)) was determined for each blood sample. These data arepresented herein as frequency percent.

Flow cytometric MN-RET data, as well as microscopy-based data, arepresented in FIG. 2. Measurements associated with the 2-color techniquewere found to be highly variable. Bivariate plots of anti-CD71-FITCversus propidium iodide fluorescence often showed events which fell on a45 degree angle, starting from the major CD71-negative population, andextending into the MN-RET quadrant. The 3-color method (with theanti-platelet immunochemical reagent) demonstrated significant numbersof events that displayed similar light scatter characteristics aserythrocytes, but unlike red blood cells, exhibited anti-CD61-PEassociated fluorescence. Exclusion of these events based on CD61expression (which requires the 3-color labeling procedure) generatedMN-RET data for days 0-4 which were much more reproducible than thoseassociated with the 2-color analyses. Interestingly, little differencewas observed between the 2- and 3-color methods for those samplescollected via heart puncture, suggesting that activated platelets and/orplatelet aggregates resulting from sub-optimal harvesting techniqueparticularly interfere with flow cytometric rodent blood MN-RET scoring.

Example 2 Human Blood Specimens, Chemotherapy/Radiation Exposure

Absolute methanol was purchased from Fisher Scientific, Springfield,N.J. (cas no. 67-56-1). Hank's balanced salt solution (HBSS), phosphatebuffered saline (PBS), and fetal bovine serum (FBS) were from MediaTechInc., Herndon, Va. Sodium heparin (cas no. 9041-08-1), RNase A (cas no.9001-99-4) and propidium iodide dye (cas no. 25535-16-4) were obtainedfrom Sigma, St. Louis, Mo. Anti-human-CD71-FITC (clone M-A712),anti-CD42b-PE (clone HIP1), and anti-rat-CD71-FITC (clone OX-26) werepurchased from BD-Pharmingen, San Diego, Calif. Fixed Plasmodiumberghei-infected rat erythrocytes (“malaria-infected rat blood”) werefrom the Rat μicroFlow® PLUS kit (Litron Laboratories, Rochester, N.Y.).

All volunteers read and signed an IRB-approved consent form. Healthyvolunteers, including the splenectomized subject, were recruited at theUniversity of Rochester Medical Center. These subjects eachcharacterized their health status as “good”, “very good” or “excellent”(as opposed to “poor” or “fair”; two current smokers were part of thisgroup: subjects hs7 and hs9 smoke 24 or fewer cigarettes per day). SeeTable 1 for other characteristics. Cancer patients were recruited fromthe Department of Radiation Oncology, James P. Wilmot Cancer Center,University of Rochester (see Table 2 below). Each healthy subjectprovided one blood sample, while cancer patients provided apre-treatment specimen and up to four additional samples drawn atapproximately 24 h intervals over the course of the first week oftherapy. Blood was obtained by standard venipuncture, and was added tomethanol fixative according to procedures described previously(Dertinger et al., “Enumeration of Micronucleated CD71-positive HumanReticulocytes with a Single-laser Flow Cytometer,” Mutat. Res. 515:3-14(2002), which is hereby incorporated by reference in its entirety).These samples were stored at −80° C. for at least 16 h. On the day ofFCM analysis, fixed blood samples were added to tubes containing HBSS.After centrifugation at 600×g, supernatants were decanted. Cells wereresuspended by striking the tubes sharply, and cells were stored on iceuntil staining and analysis (same day).

TABLE 1 Reticulocyte and micronucleated cell frequencies of healthsubjects RET^(CD71+) MN-RET^(CD71+) MN-NCE ID Sex Age (%) (%) (%) hs1 F35 0.08 0.08 0.001 hs2 F 24 0.04 0.19 0.002 hs3 M 47 0.09 0.14 0.002 hs4M 34 0.05 0.16 0.002 hs5 M 31 0.14 0.01 0.001 hs6 F 29 0.09 0.09 0.001hs7 F 48 0.08 0.07 0.001 hs8 M 39 0.05 0.06 0.001 hs9 F 44 0.51 0.020.001 hs10 F 41 0.07 0.10 0.001 hs11^(a) F 35 0.14 0.20 0.142 Average0.12 0.09 0.001 Standard 0.14 0.06 0.0005 Deviation Abbreviations: RET:young (CD71-positive) reticulocytes; MN-RET^(CD71+): micronucleatedreticulocytes (CD71-positive); MN-NCE: micronucleated normochromaticerythrocytes (CD71-negative). ^(a)Subject hs11 is splenectomized, andthese values were not included in the average and standard deviationcalculations.

TABLE 2 Cancer subject characteristics Field Size Total Bone Volume DoseID Sex Age Diagnosis Treatment (cm) Bones In Field (cm³) (Gy per day)rt1 F 72 Esophoageal cancer Radiation 14 × 25 Sternum, t-spine, part ofupper 253 1.8 ribs, and part of collar bone rt2 F 84 NCSL cancer IIaRadiation 16 × 16 Sternum, t-spine, part of upper 215 2.5 ribs, and partof collar bone rt3 M 78 NCSL cancer IV Radiation 10 × 15 Spine 103 3.0rt4 M 70 NCSL cancer IIIb Radiation 15 × 22 Sternum, t-spine, some ribs280 2.5 (especially right) rt5 M 57 Metastatic salivary Radiation   17 ×14.5 SI joints, some pelvis 255 3.5 gland rt6 M 56 Floor of mouth tumorRadiation  15 × 9.5 Jaw and c-spine 162 1.8 ch1 F 56 Small cell lungcancer 60 mg cisplatin/m²; 100 mg etoposide/m² ch2 M 75 NCSL IIIb 75 mgcisplatin/m²; 75 mg docetaxel/m² ch3 F 44 NCSL IIIb 75 mg cisplatin/m²;75 mg docetaxel/m² Abbreviations: rt: radiotherapy (megavoltageexternal-beam photon radiation delivered with linear accelerators); ch:chemotherapy; NSCL: non-small cell lung; t-spine: thoracic spine;c-spine: cervical spine; SI joints: sacral joints. Note: Total bonevolumes are approximations derived from two-dimensional X-ray film.

To prepare human blood samples for flow cytometric analysis,approximately 35 μl of fixed cells were added to polypropylene tubescontaining 100 μl of an RNase/antibody solution (850 μl HBSS with 1%FBS, 20 μl RNase A solution at 1 mg/ml, 100 μl anti-CD71-FITC, and 50 μlanti-CD42b-PE). Following successive 30 min incubations at 4° C. androom temperature, cells were washed with HBSS containing 1% FBS, and 1.6ml ice-cold propidium iodide working solution was added to each tube(1.25 μg propidium iodide/ml, HBSS as diluent). Tubes were stored at 4°C. until analysis. Dye loading was conducted for at least 10 min at 4°C., after which time cells were analyzed with a FACSCalibur flowcytometer (BD-Immunocytometry Systems, San Jose, Calif.).

At the beginning of each day of flow cytometric analysis,instrumentation and acquisition/analysis software parameters werecalibrated based on the fluorescence of a biological standard:malaria-infected rat blood. An aliquot of this blood was treated withthe same solutions used to prepare the human samples, except thatanti-rat-CD71-FITC was substituted for the anti-human immunochemicalreagent. After incubation and washing steps, cells were resuspended withpropodium iodide solution. This sample guided PMT voltage settings tooptimally resolve parasitized (MN-like) reticulocytes, and the positionof the quadrant which delineated erythrocytes with and without MN. Thehigh prevalence of reticulocytes (i.e., FITC-positive events) andmalaria-infected reticulocytes (i.e., FITC- and propidiumiodide-positive events) also helped guide compensation settings. SeeFIG. 1.

CELLQuest software v3.3 (BD-Immunocytometry Systems, San Jose, Calif.),was utilized for data acquisition and analysis. Events were triggered onthe forward scatter parameter. The gating strategy for all analyses wasbased on three regions that were designed to exclude: (1) events smalleror larger than single cells, (2) nucleated cells based on their high(2n) DNA content, and (3) platelets based on CD42b expression. See FIG.3. Data collection for each sample proceeded until the number ofCD71-positive RET (RET^(CD71+)) equaled 20,000, or when the 1.6 mlsample volume was depleted, whichever came first. The number ofRET^(CD71+), MN-RET^(CD71+), and MN-NCE was determined for each bloodsample. These data are presented herein as frequency percent.Statistical analyses were performed with JMP Software (v5, SASInstitute, Cary, N.C.). For healthy volunteers, the mean and standarddeviation for RET^(CD71+) (%), MN-RET^(CD71+) and MN-NCE (%) werecalculated. Note that the splenectomized subject's data were omittedfrom all calculations and statistical tests associated with the healthyvolunteer data-set. Cancer patients' longitudinal MN-RET^(CD71+) datawere evaluated by least squares regression (chemotherapy patients' datawere pooled and evaluated separately from radiotherapy patients' data).Based on r² values a polynomial curve of degree 3 and 2 were chosen tomodel the chemo- and radiotherapy MN-RET^(CD71+) time-course data,respectively. ANOVA tables which accompany the JMP program's regressionanalyses partitioned the total variation into components, and comparedthe best-fit curves to a simple mean response model. A P value <0.05 wasused to indicate a significant regression effect (i.e., time-dependenttrend). Additionally, all cancer patients' initial (before treatment)MN-RET^(CD71+) frequencies were compared to healthy volunteers'MN-RET^(CD71+) values using a two-tailed Student's t-test (significanceindicated by P<0.05).

The staining procedure utilized for these studies resulted influorescent resolution of the target MN-RET population. Malaria-infectedrat blood provided cells which mimic micronucleated erythrocytes well,and their prevalence and uniform staining characteristics were valuablefor calibrating flow cytometer settings between days of analysis. Theseattributes also provided a means for rationally setting the position ofthe quadrant used to define the human erythrocyte subpopulations ofinterest (see FIG. 4A-B).

The results from healthy volunteers are presented in Table 1 above.Based on the average RET^(CD71+) (%) value for these subjects, and alsoon analyses whereby reticulocyte frequencies were measured based onRNA-associated fluorescence, we estimate that the anti-CD71-FITC reagentlabeled approximately the youngest 10% of RNA-positive RET (see FIG. 5).For eusplenic subjects, this young cohort of erythrocytes exhibited anaverage value of 0.09% MN-RET^(CD71+). The efficiency by which the humanspleen removes MN from circulation was demonstrated by the extremely lowvalues observed for these healthy subjects' mature erythrocytes(0.001-0.002% MN-NCE). In addition to these samples, blood from asplenectomized but otherwise healthy individual was analyzed (subjecths11; MN-RET^(CD71+)=0.20%).

As expected, chemo- and radiotherapy reduced the frequency ofRET^(CD71+) over the course of cancer treatment (see FIG. 6). Theproportion of red marrow space that was subjected to treatment waslikely an important determinant for the range of responses observed. Forinstance, chemotherapy subjects, who presumably received systemicexposure, showed the greatest reduction to RET^(CD71+) (%). In fact, inthe case of subject ch2, treatment-related reduction to peripheral bloodRET^(CD71+) was so severe as to preclude an accurate determination ofMN-RET^(CD71+) frequency three and four days post-treatment (RET^(CD71+)(%)=0.01).

Regarding cancer patients' MN-RET^(CD71+) frequencies, no significantdifference was observed between pre-treatment values and those of thehealthy volunteers. However, as illustrated by FIG. 6, the majority ofcancer patients demonstrated elevated MN-RET^(CD71+) frequencies overthe course of therapy. Regression analyses indicate that thesetime-dependent increases in MN-RET^(CD71+) are statistically significant(P=0.0166 and 0.0081 for pooled chemo- and radiotherapy patients,respectively).

As with the RET^(CD71+) population, MN-RET^(CD71+) frequencies werelikely influenced by the proportion of red marrow space exposed. Thus,in the case of radiotherapy, MN-induction was muted to the extent thatother (non-exposed) sites of erythropoiesis supplied the peripheralblood compartment with MN-RET^(CD71+) at a baseline frequency. Forinstance, the little or no change in MN-RET^(CD71+) (%) for subjects rt3(spine irradiation) and rt6 (jaw/spine irradiation) is likely related tothe low proportion of active hematopoietic red marrow which was exposed(as reflected by a lack of change in RET^(CD71+) (%)). Conversely, thehigher MN responses observed for patients undergoing chemotherapy orlarge field chest irradiation was likely due to the large amounts of redmarrow exposure that was achieved. In addition to exposure fieldlocation/size, another factor which may help explain modest or noobserved effects for subjects rt1 and rt6 is a relatively lowerradiation intensity (1.8 Gy per day). A simple model which describes themajor variables which appear to affect peripheral blood MN-RET^(CD71+)frequency is illustrated by FIG. 7.

Data presented herein support the concept that the incidence of MN-RETin human peripheral blood circulation can be used to index recentcytogenetic damage. Increased MN-RET^(CD71+) values were evident 2-4days after initiation of treatment, and this is in agreement with thekinetics of erythroblast differentiation and the entry of newly formederythrocytes into the peripheral blood compartment (Hillman and Finch,“Erythropoiesis: Normal and Abnormal,” Semin. Hematol. 4:327-336 (1967),which is hereby incorporated by reference in its entirety). This is madepossible by an analytical system which is capable of restrictinganalyses to the most immature fraction of RET. The rarity ofRET^(CD71+), coupled with the low frequency of MN events, makes thehigh-throughput nature of the scoring system an essentialcharacteristic. For instance, the time required to collect data on theflow cytometer was approximately 25 min per sample. Even with theserelatively lengthy data acquisition times, the number of RET^(CD71+)interrogated for MN per sample was typically about 10,000. Methods forenriching blood for newly formed erythrocytes have been described in theliterature (Abramsson-Zetterberg et al., “Human CytogeneticBiomonitoring Using Flow-Cytometric Analysis of Micronuclei inTransferrin-Positive Immature Peripheral Blood Reticulocytes,” Environ.Mol. Mutagen. 36:22-31 (2000); Choy and MacGregor, “Density-gradientEnrichment of Newly-Formed Mouse Erythrocytes: Application to theMicronucleus Test,” Mutat. Res. 130:159-164 (1984), which is herebyincorporated by reference in its entirety), and these could potentiallylower flow cytometer data acquisition time, and also increase thenumbers of RET interrogated. However, an objective of the presentinvention has been to establish a method that requires as fewmanipulations with whole, unfixed human blood as possible. That is, apriority was placed on keeping the procedure simple and reproducible forthe medical technologist in the clinical environment.

The simplicity of the processing steps may be of practical importancefor using the technique in any clinical and/or biomonitoringapplications. The invention described herein addresses the relativelylengthy flow cytometry data acquisition times associated flow cytometricanalyses that do not include a physical RET enrichment scheme. Instead,a technique for improving sample throughput capabilities is demonstratedin Example 3 below.

The blood samples from 10 healthy volunteers were important forestimating baseline RET^(CD71+), MN-RET^(CD71+), and MN-NCE values. Theaverage frequency of MN-RET^(CD71+) was similar, although somewhatlower, than values observed in the bone marrow or in the peripheralblood circulation of splenectomized human subjects (0.09% compared toapproximately 0.2-0.3%; see (Goetz et al., “Relationship BetweenExperimental Results in Mammals and Man: Cytogenetic Analysis of BoneMarrow Injury Induced by a Single Dose of Cyclophosphamide,” Mutat. Res.31:247-254 (1975); Krogh Jensen and Nyfors, “Cytogenetic Effect ofMethotrexate on Human Cells In Vivo,” Mutat. Res. 64:339-343 (1979); Abeet al., “Micronuclei in Human Bone Marrow Cells: Evaluation of theMicronucleus Test Using Human Leukemia Patients Treated withAntileukemic Agents,” Mutat. Res. 130:113-120 (1984); Schlegel et al.,“Assessment of Cytogenetic Damage by Quantitation of Micronuclei inHuman Peripheral Blood Erythrocytes,” Cancer Res. 46:3717-3721 (1986);Smith et al. (“Micronucleated Erythrocytes as an Index of CytogeneticDamage in Humans: Demographic and Dietary Factors Associated withMicronucleated Erythrocytes in Splenectomized Subjects,” Cancer Res.50:5049-5054 (1990); and MacGregor et al., “Spontaneous Genetic Damagein Man: Evaluation of Interindividual Variability, Relationship AmongMarkers of Damage, and Influence of Nutritional Status,” Mutat. Res.377:125-135 (1997), each of which is hereby incorporated by reference inits entirety). This is likely related to erythrophagocytosis activity,which may not be fully negated by restricting analyses to RET^(CD71+).Even so, when compared to MN-NCE values (0.002%), the averageMN-RET^(CD71+) frequency of 0.09% provides evidence that the analyticalsystem described herein does effectively minimize the impact that spleenfunction has on peripheral blood MN frequency.

Similar to Smith et al. (“Micronucleated Erythrocytes as an Index ofCytogenetic Damage in Humans: Demographic and Dietary Factors Associatedwith Micronucleated Erythrocytes in Splenectomized Subjects,” CancerRes. 50:5049-5054 (1990), which is hereby incorporated by reference inits entirety), a greater than 10-fold range of MN-RET^(CD71+)frequencies in presumably healthy volunteers was observed. Knowledge ofthis extent of variation in spontaneous MN-RET^(CD71+) frequency wasvaluable information for designing experiments to evaluategenotoxicant-induced MN. That is, these data clearly indicated thedesirability of obtaining pre-treatment blood samples when studyingoncology patients. As with the healthy subjects, pre-treatmentMN-RET^(CD71+) frequencies of cancer patients were indeed variable(range=0.02-0.17%). Even so, pre-treatment samples served aspatient-specific controls, and were helpful for assessingtreatment-related changes to MN frequency in the relatively small numberof subjects studied.

For the present study, cancer patients' blood samples were used for theexpress purpose of evaluating the FCM-based scoring systems' ability todetect MN induced by known physical and chemical genotoxic agents. Manyother reports exist in which the micronucleus endpoint has been measuredin cancer patients. Since the clinical efficacy of ionizing radiationand the majority of antineoplastic drugs has most often been attributedto their ability to cause irreparable DNA damage, MN formation has beenevaluated as a nonclonogenic endpoint that might provide valuablepatient-specific information regarding sensitivity to treatment(Bhattathiri et al., “Serial Cytological Assay of MicronucleusInduction: A New Tool to Predict Human Cancer Radiosensitivity,”Radiother. Oncol. 41:139-142 (1996); and Guo et al., “A SignificantCorrelation Between Clonogenic Radiosensitivity and the SimultaneousAssessment of Micronucleus and Apoptotic Cell Frequencies,” Int. J.Radiation Biol. 75:857-864 (1999), each of which is hereby incorporatedby reference in its entirety). Other reports have suggested that theendpoint may have prognostic value (Zolzer et al., “Changes in S-phaseFraction and Micronucleus Frequency as Prognostic Factors inRadiotherapy of Cervical Carcinoma,” Radiother. Oncol. 36:128-132(1995); Widel et al., “The Increment of Micronucleus Frequency inCervical Carcinoma During Irradiation In Vivo and Its Prognostic Valuefor Tumor Radiocurability,” Br. J. Cancer 80:1599-1607 (1999); Widel etal., “Micronucleus Assay In Vivo Provides Significant PrognosticInformation in Human Cervical Carcinoma: The Updated Analysis,” Int. J.Radiat. Biol. 77:631-636 (2001), each of which is hereby incorporated byreference in its entirety), or that it may be valuable for detectingpredisposition to certain cancers (Doneda et al., “High SpontaneousChromosome Damage in Lymphocytes From Patients With HereditaryMegaduodenum,” Mutat. Res. 348:33-36 (1995); Berg-Drewniok et al.,“Increased Spontaneous Formation of Micronuclei in Cultured Fibroblastsof First-degree Relatives of Familial Melanoma Patients,” Cancer Genet.Cytogenet. 97:106-110 (1997); Scott et al., “Radiation-inducedMicronucleus Induction in Lymphocytes Identifies a High Frequency ofRadiosensitive Cases Among Breast Cancer Patients: A Test forPredisposition?” Br. J. Cancer 77:614-620 (1998); Burrill et al.,“Heritability of Chromosome Radiosensitivity in Breast Cancer Patients:A Pilot Study with the Lymphocyte Micronucleus Assay,” Int. J. Radiat.Biol. 76:1617-1619 (2000), each of which is hereby incorporated byreference in its entirety). For these various studies, MN have beenstudied in tumor biopsy material, as well as in blood lymphocytes whichhave been stimulated to divide in culture (Fenech, “TheCytokinesis-block Micronucleus Technique: A Detailed Description of theMethod and its Application to Genotoxicity Studies in HumanPopulations,” Mutat. Res. 285:35-44 (1993), each of which is herebyincorporated by reference in its entirety).

Based on data reported herein, CD71-positive RET in peripheral bloodcirculation represent an alternate target cell population which can beused to assess DNA damaging activity. Analyses based on these cellsoffer several advantages, including: minimally invasive cell harvest;low blood volume requirement; simple fixation/staining procedures; andunbiased, automated scoring. MN-RET^(CD71+) measurements may proveuseful to researchers and clinicians who are involved in cancersusceptibility testing, prognosis, or treatment optimization.Additionally, these measurements may represent a minimally invasivebiomonitoring tool for assessing occupational, environmental, ornutritional factors that might be expected to have genotoxicconsequences (MacGregor et al., “Spontaneous Genetic Damage in Man:Evaluation of Interindividual Variability, Relationship Among Markers ofDamage, and Influence of Nutritional Status,” Mutat. Res. 377:125-135(1997); Anwar et al., “Chromosomal Aberrations and MicronucleusFrequency in Nurses Occupationally Exposed to Cytotoxic Drugs,”Mutagenesis 9:315-317 (1994); Ilyinskikh et al., “Micronucleus Test ofErythrocytes and Lymphocytes in the Blood of the People Living in theRadiation Pollution Zone as a Result of the Accident at the SiberianChemical Plant on Apr. 6, 1993,” Mutat. Res. 36:173-178 (1996); Maffeiet al., “Micronuclei Frequencies in Hospital Workers OccupationallyExposed to Low Levels of Ionizing Radiation: Influence of Smoking Statusand Other Factors,” Mutagenesis 17:405-409 (2002); Fenech, “Biomarkersof Genetic Damage for Cancer Epidemiology,” Toxicology 181-182:411-416(2002), each of which is hereby incorporated by reference in itsentirety).

Example 3 High Speed MN-RET Data Acquisition

The Informed consent was obtained from a small cell lung cancer patientwho was recruited from the James P. Wilmot Cancer Center, University ofRochester. The cancer patient provided a blood sample just prior to, andagain three days after treatment with 60 mg cisplatin/m² and 100 mgetoposide/m². Blood was obtained by standard venipuncture, and was addedto methanol fixative according to the present invention. Fixed sampleswere stored at −80° C. for at least one day before flow cytometricanalysis.

Fixed human blood specimens (1-2 ml) were added to tubes containing 12ml ice-cold HBSS and cells were collected by centrifugation.Supernatants were decanted and pellets were tapped loose. For 2-colorlabeling, 35 μl cells were added to polypropylene tubes containing 100μl of an RNase/antibody solution (900 μl HBSS with 1% FBS, 100 μlanti-CD71-FITC, and RNase A at 20 μg/ml). Following successive 30 minuteincubations at 4° C. and room temperature, cells were washed with 5 mlHBSS containing 1% FBS, and finally resuspended in 1.5 ml propidiumiodide solution. Stained samples were stored at 4° C. until analysis(same day). For 3-color analyses, similar procedures were used, exceptthat 50 μl anti-CD42b-PE replaced 50 μl of HBSS with 1% FBS in theRNase/antibody solution. Also, for the alternative “highdensity/FL1-thresholding technique”, washed cells were concentrated withvigorous decanting after the initial centrifugation step, and entirecell pellets were added to polypropylene tubes containingRNase/antibodies. By high density, it is believed that the resultingcell concentration achieved is about 80 million cells/ml. This isroughly 15 times greater than the cell density utilized in the precedingexamples.

At the beginning of each day of flow cytometric analysis,instrumentation and acquisition/analysis software parameters werecalibrated based on the fluorescence of a biological standard:malaria-infected mouse or rat blood. An aliquot of this blood wasincubated with the same RNase, anti-CD71-FITC, and propidium iodidesolutions used for test samples (except that anti-rat-CD71-FITC wassubstituted for the anti-human immunochemical reagent). As describedpreviously, these samples guided PMT voltage and electronic compensationsettings to optimally resolve parasitized (MN-like) reticulocytes, andalso guided the position of the quadrant which delineated erythrocyteswith and without MN.

Data acquisition and analyses were performed using a FACSCalibur flowcytometer providing 488 nm excitation, running CellQuest software(v3.3). Anti-CD71-FITC, anti-platelet-PE, and propidium iodidefluorescence signals were detected in the FL1, FL2, and FL3 channels,respectively. Unless otherwise stated, events were triggered with a FSCthreshold so that all cell-sized events were collected. For human bloodsamples analyzed according to the high density/FL1-thresholdingtechnique, events were triggered using an FL1 threshold which eliminatedCD71-negative erythrocytes (NCE) from consideration. The stop mode wasset so that 20,000 CD71-positive reticulocytes were analyzed for MN persample.

Triplicate blood specimens were analyzed according to 2- and 3-colorlabeling procedures as described above. MN-RET values for the 3-colormethod were consistently lower than those for corresponding 2-coloranalyses (FIG. 8). These quantitative differences suggested thatalthough these specimens were collected via arm venipuncture, and hencewould not be expected to have large numbers of activated platelets,platelets none-the-less interfered with MN-RET measurements. Aside fromthe difference observed between 2- and 3-color MN-RET values,fluorescence microscopy confirmed the identity of CD42b-positive eventsas platelets and platelet aggregates.

The specimens from this chemotherapy patient were further analyzedaccording to the 3-color labeling scheme, but at very high celldensities. These extreme cell densities would ordinarily be above theFACSCalibur's 7,000 events per second maximum rate. This was addressedby changing from a FSC to an FL1 trigger, and by adjusting the thresholdso that only CD71-positive erythrocytes were evaluated. Thismodification generated MN-RET values that were in good agreement withthose produced with the FSC threshold/lower density analyses (3-color).The main benefit of the high density technique was that the average timeto interrogate 20,000 RET per sample was reduced from approximately 24minutes on average to less than 4 minutes. Furthermore, the size of thedata files was reduced from ≧105 Mb to less than 1 Mb. FIG. 8.

Example 4 Assessment of Splenic Filtration Function

Discarded EDTA-blood specimens from unselected children with documentedHbSS or HbSC disease were fixed according to methods of the presentinvention. Codes specimens were shipped to Litron Laboratories on dryice for flow cytometric analysis.

Fixed blood specimens (2 ml) were combined with 12 ml ice-cold HBSS andcells were collected by centrifugation. Supernatants were decanted andpellets were tapped loose. The 3-color labeling procedure was utilizedwhereby approximately 35 μl of washed cells were added to polypropylenetubes containing 100 μl of an RNase/antibody solution (850 μl HBSS with1% FBS, 100 μl anti-CD71-FITC, 50 μl anti-CD42b-PE, and RNase A at 20μg/ml). Following successive 30 minute incubations at 4° C. and roomtemperature, cells were washed with 5 ml HBSS containing 1% FBS, andfinally resuspended in 1.5 ml propidium iodide solution. Stained sampleswere stored at 4° C. until analysis (same day).

At the beginning of each day of flow cytometric analysis,instrumentation and acquisition/analysis software parameters werecalibrated based on the fluorescence of a biological standard:malaria-infected mouse or rat blood. An aliquot of this blood wasincubated with the same RNase, anti-CD71-FITC, and propidium iodidesolutions used for test samples (except that anti-rat-CD7′-FITC wassubstituted for the anti-human immunochemical reagent). As describedpreviously, these samples guided PMT voltage and electronic compensationsettings to optimally resolve parasitized (MN-like) reticulocytes, andalso guided the position of the quadrant which delineated erythrocyteswith and without MN.

Data acquisition and analyses were performed using a FACSCalibur flowcytometer providing 488 nm excitation, running CellQuest software(v3.3). Anti-CD71-FITC, anti-platelet-PE, and propidium iodidefluorescence signals were detected in the FL1, FL2, and FL3 channels,respectively. Events were triggered with a FSC threshold so that allcell-sized events were collected. The stop mode was set so that1,000,000 erythrocytes were analyzed for MN per sample.

The frequency of MN-NCE for pediatric patients with documented HbSS andHbSC disease are presented in FIGS. 9A-B, respectively. HbSS patientsranged in age from 0.2 to 17.1 years, and would be expected to providevarying degrees of accumulated vaso-occlusive damage to the spleen. Asexpected, an age-dependent increase in HJB values for HbSS patients isstatistically significant (p=0.0113, linear regression analysis, JMPsoftware v5). On the other hand, specimens from 12 HbSC patients did notexhibit a significant age-dependent effect on MN-NCE values. In fact,for ten of twelve HbSC patients, MN-NCE values are in the same range asthose observed in healthy volunteers (less than 100×10⁻⁶). Takentogether, these 43 specimens lend support the premise that MN-NCEmeasurements provided by the present invention are indicative of splenicerythrophagocytosis function, and that there may be prognostic value tothese measurements, as the HbSS genotype is known to have a more severeclinical course relative to HbSC disease. Thus, it is likely thatmultivariate models designed to predict sickle cell disease severitywould benefit from these spleen function data.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

1. A method for the enumeration of micronucleated erythrocytepopulations while excluding false-positive micronuclei scoring eventscaused by platelet and platelet-associated aggregates, the methodcomprising: providing a sample comprising erythrocyte populationsincluding mature normochromatic erythrocytes, reticulocytes,micronucleated normochromatic erythrocytes, micronucleatedreticulocytes, or combinations thereof, with the erythrocyte populationsbeing in suspension and substantially free of aggregates, permeable to anucleic acid dye and RNase, with cell surface markers in a formrecognizable by an antibody, and able to exhibit substantially lowautofluorescence; substantially degrading RNA of reticulocytes in thesample with RNase; contacting the sample with a first reagent comprisinga fluorescent label and having binding specificity for a surface markerfor reticulocytes and with a second reagent comprising a fluorescentlabel and having binding specificity for a surface marker for platelets,wherein the fluorescent emission spectrum of the fluorescent labels ofthe first and second reagents do not substantially overlap; stainingcellular DNA with a nucleic acid staining dye having a fluorescentemission spectrum which does not substantially overlap with thefluorescent emission spectrum of the fluorescent labels of the first andsecond reagents; exciting the nucleic acid staining dye, the fluorescentlabel associated with the reticulocytes, and the fluorescent labelassociated with platelets using light of appropriate excitationwavelength for both the nucleic acid staining dye and the fluorescentlabels to produce fluorescent emission; and detecting the fluorescentemission and light scatter produced by the erythrocyte populations andplatelets, and counting the number of cells from one or more erythrocytepopulations in said sample while excluding scoring events caused byplatelets and platelet-associated aggregates from scoring events formicronucleated erythrocytes.
 2. The method according to claim 1 whereinthe total cellular concentration of the sample is greater than about 30million cells/ml.
 3. The method according to claim 2 wherein saidcounting is carried out only for reticulocytes.
 4. The method accordingto claim 1 wherein said providing comprises: obtaining a peripheralblood or bone marrow sample from a mammal; and treating the obtainedsample with a reagent that renders the erythrocytes and reticulocytespermeable, and thereby affords the provided sample.
 5. The methodaccording to claim 4 further comprising: administering a DNA-damagingagent to the mammal prior to said obtaining.
 6. The method according toclaim 5 further comprising: administering a suspected protective agentthat protects against DNA damage to the mammal simultaneous orcontemporaneous with administration of the DNA-damaging agent; andmeasuring any protective effect provided by the suspected protectiveagent.
 7. The method according to claim 4, wherein the reagent thatrenders the erythrocytes and reticulocytes permeable is an alcohol at atemperature of from about −40° C. to about −90° C.
 8. The methodaccording to claim 7 wherein the alcohol is a primary alcohol or asecondary alcohol.
 9. The method according to claim 1 furthercomprising: removing first and second reagents not bound to cells in thecontacted sample.
 10. The method according to claim 9 wherein saidremoving comprises: washing the contacted sample and exposing the washedsample to centrifugal forces sufficient to separate unbound first andsecond reagents from first and second reagents bound to cells.
 11. Themethod according to claim 9 wherein said removing is carried out priorto said staining with nucleic acid dye.
 12. The method according toclaim 1 wherein the surface marker for reticulocytes is CD71.
 13. Themethod according to claim 1 wherein the surface marker for platelets isCD9, CDw17, CD29, CD31, CD32, CD41, CD42a, CD42b, CD42c, CD42d, CD43,CD46, CD49f, CD51, CD60a, CD61, CD62P, CD63, CD69, CD82, CD98, CD102,CD110, CD112, CDw119, CD120a, CD128a, CD128b, CD130, CD132, CD140a,CD141, CD148, CD151, CD165, CD184, CD226, or CD245.
 14. The methodaccording to claim 1 wherein said exciting and detecting are carried outusing a flow cytometer comprising either a single laser or two or morelasers.
 15. The method according to claim 14 further comprising:calibrating the flow cytometer using a biological standard which hasbeen treated in parallel with the sample.
 16. The method according toclaim 15 wherein said calibrating comprises setting photomultiplier tubevoltage, setting electronic compensation parameters, defining theposition of regions that distinguish a micronucleus-containingerythrocyte from a mature normochromatic erythrocyte or a reticulocyte,and combinations thereof.
 17. The method according to claim 15 whereinthe biological standard is an erythrocyte sample obtained from amalaria-infected mammal.
 18. The method according to claim 1 whereinsaid substantially degrading and said contacting are carried outsimultaneously.
 19. The method according to claim 1 wherein the mammalis a human, monkey, chimpanzee, rat, mouse, or beagle.
 20. The methodaccording to claim 1 further comprising: administering a chemical agentto a mammalian subject prior to said providing, wherein a statisticallysignificant difference in the percentage of micronucleatednormochromatic erythrocytes and/or micronucleated reticulocytes from abaseline micronucleated normochromatic erythrocyte and/or micronucleatedreticulocyte value in unexposed mammals indicates the genotoxicpotential of the chemical agent.
 21. The method according to claim 20wherein the chemical agent is a pharmaceutical.
 22. The method accordingto claim 1 further comprising: administering a DNA-damaging agent to amammalian subject prior to said providing, wherein a statisticallysignificant difference in the percentage of micronucleated reticulocytesfrom a baseline micronucleated reticulocyte value in similarly exposedmammals that possess normal sensitivity to the DNA-damaging agentindicates the hypersensitivity or insensitivity of the mammalian subjectto the DNA-damaging agent.
 23. The method according to claim 22 whereinthe DNA-damaging agent is a physical DNA damaging agent or a chemicalDNA damaging agent.
 24. The method according to claim 1, wherein theperipheral blood or bone marrow samples are obtained from mammalsexposed to one or more suspected DNA-damaging agents in an environment,and wherein a statistically significant increase in the percentage ofmicronucleated reticulocytes from a baseline micronucleated reticulocytevalue in unexposed mammals indicates that the environment contains oneor more DNA-damaging agents.
 25. The method according to claim 24wherein the one or more suspected DNA-damaging agents are physical DNAdamaging agents, chemical DNA damaging agents, or combinations thereof.26. The method according to claim 1 further comprising: administering asuspected modulating agent that may modify endogenously or exogenouslyinduced genetic damage to a mammalian subject prior to said providing,wherein a statistically significant difference in the percentage ofmicronucleated reticulocytes from a baseline micronucleated reticulocytevalue in mammals which are similarly treated except for the suspectedmodulating agent indicates that the agent can modify endogenously orexogenously induced DNA damage.
 27. The method according to claim 26wherein said administering is carried out simultaneously orcontemporaneously with endogenously induced DNA damage.
 28. The methodaccording to claim 26 further comprising: exposing the mammalian subjectto an exogenous DNA-damaging agent prior to said providing.
 29. Themethod according to claim 28 wherein said administering is carried outsimultaneously or contemporaneously with said exposing.
 30. The methodaccording to claim 1 further comprising: subjecting a mammal to apredetermined diet or a dietary nutrient that may modify endogenously orexogenously induced DNA damage prior to said providing, wherein (i) astatistically significant difference in the percentage of MN-RET frombaseline MN-RET values in unexposed mammals indicates that the diet ordietary nutrient can modify endogenous DNA damage; or (ii) astatistically significant difference in the percentage of MN-RET fromMN-RET values in mammals treated with the same genotoxicant but withoutthe predetermined diet or the dietary nutrient indicates that the dietor the dietary nutrient can modify exogenously induced DNA damage. 31.The method according to claim 30 wherein said subjecting is carried outsimultaneously or contemporaneously with endogenously induced DNAdamage.
 32. The method according to claim 30 further comprising exposingthe mammal to an exogenous DNA damaging agent.
 33. The method accordingto claim 32 wherein said subjecting is carried out simultaneously orcontemporaneously with said exposing.